Background: Many activator proteins function by directly contacting RNA polymerase (RNAP), however the exact effects of these interactions are generally not understood. The long-range goal of this project is to elucidate the structure of the transcription activator protein RhaR and to relate this structure to the molecular mechanisms used by RhaR to activate transcription. In the presence of L-rhamnose, RhaR activates expression of the Escherichia coli rhaSR operon. Full activation of rhaSR expression also requires the CRP protein. The central hypothesis is that specific interactions between RhaR, CRP and RNAP are required for transcription activation. Structural information for RhaR will enable a more detailed understanding of these interactions. Aims" 1) Structural analysis of RhaR protein; 2) Characterize RhaR interactions with RNAP; and 3) Characterize RhaR interactions with CRP. A major goal will to obtain enough soluble RhaR protein to use X-ray crystallography to determine its structure. One approach will be to identify refolding conditions for a highly overexpressed, but insoluble, RhaR-His6 fusion protein. We will also express the individual RhaR domains, which may be more soluble than intact RhaR, and which certainly will be more readily refolded if they are insoluble. Finally, we will: test whether co-expression of GroEL improves the yield of soluble RhaR; follow up on our evidence that RhaR may be sensitive to inactivation by oxidation; and use the low-concentration purified RhaR that we have in hand to identify optimal conditions for RhaR. The other goal of the proposal is to characterize the interactions that RhaR makes with RNAP and CRP, and to correlate this functional information with the structure. We will biochemically characterize two contacts between RhaR and the RNAP sigma70 subunit that we have genetically identified. We will also test whether RhaR interacts with the RNAP ft subunit Cterminal domain, and if so biochemically characterize these interactions. We will determine whether direct contacts between RhaR and CRP contribute to the apparent ability of RhaR to influence the DNA sites in the rhaSR promoter to which CRP binds. Significance: The finding that many regions of the crystal structures of a bacterial and a yeast RNAP can be virtually superimposed suggests that they may utilize similar mechanisms for interaction with activators. Also, RhaR is a member of the large AraC/XylS family of activators, many of which activate virulence factors in bacterial pathogens and are of interest as antibacterial targets. Understanding the mechanisms used by AraC/XylS family members to activate transcription will be important for rational design of anti-AraC/XylS family agents.